John E. Battersby

Sustained release of recombinant human growth hormone from dextran via hydrolysis of an imine bond

Dextran T40 was oxidized with periodate to form aldehyde groups to which recombinant human growth hormone (rhGH) was attached. Complex formation involved the reversible formation of an imine conjugate and the extent of complex formation was proportional to the degree of oxidation of the dextran. The complex was characterized by a variety of techniques including SEC, SDS-PAGE and IEF. In vitro, the rate of release of rhGH from a rhGH-dextran complex was inversely proportional to the degree of oxidation of the dextran. The released protein was characterized by peptide mapping, N-terminal sequencing, SEC and SDS-PAGE. Sustained release of rhGH was observed in vivo, based on the observation of significant weight gain in a rat bioassay.

Analytical Characterization of Monoclonal Antibodies: Linking Structure to Function

The basic structural features of antibodies, including their primary structures (Edelman et al. 1969), were established well before the concept of therapeutic antibodies was conceived. These molecules are now known to bear multiple sources of microheterogenity that can have a dramatic effect on in vivo and in vitro properties. Rituximab (Rituxan®), Trastuzumab (Herceptin®) and omalizumab (Xolair®) are three examples of therapeutic IgG1/kappa subclass antibodies produced by Genentech, Inc.; these molecules are the main subject of this discussion on the impacts of common and unique antibody modifications on functional properties.

Application of capillary high-performance liquid chromatography to biotechnology, with reference to the analysis of recombinant DNA-derived human growth hormone

Using capillary HPLC, femtomole amounts of recombinant DNA-derived human growth hormone (rhGH) have been successfully detected from solutions at nanomolar concentrations. The separation used capillaries of 15 cm x 320 microns I.D. and detection was with a UV absorbance detector containing a capillary Z-shaped flow-cell. A sample of rhGH that was recovered from rat serum was analyzed by capillary reversed-phase HPLC, using both acidic- and neutral-pH mobile phases, as well as by capillary ion-exchange chromatography. When compared to HPLC separations performed at flow-rates of 1 ml/min, the sensitivity of the detection was increased 200 times, without any loss in resolution. Sub-microgram amounts of rhGH were also analyzed by tryptic mapping using capillary HPLC and peptides were identified by capillary LC-MS.

Purification and quantitation of tumor necrosis factor receptor immunoadhesin using a combination of immunoaffinity and reversed-phase chromatography

The development of an automated, dual column assay to quantitate and recover the glycoprotein, tumor necrosis factor receptor immunoadhesin (TNFr-IgG) from monkey plasma, human serum, cell culture fluid and buffer samples is described. A combination of immunoaffinity and reversed-phase chromatographies are used. The targeted protein was captured using an anti-TNFr-1 monoclonal antibody immobilized on POROS resin. After non-specific adsorption had been reduced, the affinity column was placed in-line with a reversed-phase column and eluted with dilute acid. The reversed-phase column was subsequently eluted with an acetonitrile gradient and the TNFr-IgG collected and quantitated by comparison with peak areas of similarly treated standards. Detection was performed by measurement of absorbance at 214 nm. The dynamic range is from 0.5-15 microg total sample. Samples were quantitated and recovered from monkey and human pharmacokinetics samples, as well as from cell culture fluid and buffers. The lowest concentrations assayed were 100 ng ml(-1). Quantitation is reproducible, with a coefficient of variation of 2%. The procedure was used to develop a pharmacokinetic profile for the clearance of TNFr-IgG in humans and cynomolgus monkeys. Sufficient material was recovered such that the glycoforms could be identified. Additionally it has been used for process monitoring. The results compared favorably with data generated by ELISA. Optimization of the method and results are presented.